Apoptosis, or programmed cell death, is a naturally occurring process that has been strongly conserved during evolution to prevent uncontrolled cell proliferation. This form of cell suicide plays a crucial role in the development and maintenance of multicellular organisms by eliminating superfluous or unwanted cells. However, if this process goes awry, excessive apoptosis results in cell loss and degenerative disorders including neurological disorders such as Alzheimers, Parkinsons, ALS, retinitis pigmentosa and blood cell disorders, while insufficient apoptosis contributes to the development of cancer, autoimmune disorders and viral infections (Thompson, Science, 1995, 267, 1456-1462).
Several stimuli can induce apoptosis, and recently, major advances have been made in understanding the signaling pathways mediated by the cell surface cytokine receptors activated by these stimuli, TNFR-1 and CD95 (Fas/APO-1).
The pathways leading from these receptors involve a proteolytic cascade orchestrated by a family of enzymes known as caspases (Thornberry, Br. Med. Bull., 1997, 53, 478-490). The most upstream caspase identified to date is caspase 8 (also known as CAP4, FLICE, MACH and Mch5). Caspase 8 is ubiquitously expressed in both fetal and adult tissues, with the exception of fetal brain and when overexpressed, induces apoptosis (Muzio et al., Cell, 1996, 85, 817-827). Therefore, it is currently believed that modulation of caspase 8 expression represents a potential therapeutic target in a variety of deregulated apoptotic pathologic conditions.
Caspase 8 interacts with the CD95 receptor in association with the adapter protein, FADD, through a previously identified protein motif contained within both proteins known as the death domain (Muzio et al., Cell, 1996, 85, 817-827). Once recruited to the death-inducing signaling complex (DISC), caspase 8 undergoes autoproteolytic cleavage and subsequent activation (Martin et al., J. Biol. Chem., 1998, 273, 4345-4349; Medema et al., Embo J., 1997, 16, 2794-2804; Muzio et al., J. Biol. Chem., 1998, 273, 2926-2930; Srinivasula et al., Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 14486-14491). While downstream effector caspases have been shown to cleave several classes of protein substrates, having somewhat redundant roles, upstream caspases such as caspase 8 function primarily to cleave and activate caspases downstream of receptor activation. One exception is cytosolic phospholipase A2. Caspase 8 has recently been shown to cleave this non-caspase proinflammatory enzyme (Luschen et al., Biochem. Biophys. Res. Commun., 1998, 253, 92-98).
It has recently been demonstrated that caspase 8 can be activated by several death receptor-independent pathways as well. Caspase 8 can be activated by anticancer drugs in the absence of CD95 receptor activation in human leukemic T-cell lines suggesting the presence of an alternate apoptotic pathway (Bantel et al., Cancer Res., 1999, 59, 2083-2090; Wesselborg et al., Blood, 1999, 93, 3053-3063). Medema et al. have also demonstrated that caspase 8 is cleaved by granzyme B in HeLa cells, indicating its involvement in perforin-induced apoptosis, another CD95-independent apoptotic pathway (Medema et al., Eur. J. Immunol., 1997, 27, 3492-3498). Other CD95-independent pathways include mediation by nitric-oxide (Chlichlia et al., Blood, 1998, 91, 4311-4320), cytochrome c (Kuwana et al., J. Biol. Chem., 1998, 273, 16589-16594), and the Sendai virus (Bitzer et al., J. Virol., 1999, 73, 702-708).
Caspase 8 represents a potential therapeutic target in several diseases including AIDS and AIDS-related disorders. It has been shown to be upregulated by the AIDS viral Tat protein (Bartz and Emerman, J. Virol., 1999, 73, 1956-1963; Peter et al., Br. Med. Bull., 1997, 53, 604-616). Mandruzzato et al. identified an antigen recognized by cytolytic T lymphocytes encoded by a mutated form of the caspase 8 gene in head and neck carcinoma cells. This mutation, found only in the tumor cells, alters the stop codon thereby adding 88 amino acids to the protein and reducing the activity of the caspase (Mandruzzato et al., J. Exp. Med., 1997, 186, 785-793).
A number of alternatively spliced isoforms of caspase 8 have been identified with varying activity (Scaffidi et al., J. Biol. Chem., 1997, 272, 26953-26958). Disclosed in U.S. Pat. No. 5,837,837 and PCT publication WO 98/38200 are the nucleic acid sequences encoding caspase 8h and 8i as well as vectors containing nucleic acid molecules encoding these isoforms and host cells containing these vectors. Also disclosed are antibodies, transgenic animals, and a method for treating a patient with a compound that modulates the expression or activity of these isoforms (Hunter et al., 1998; Hunter et al., 1998).
Disclosed in U.S. Pat. Nos. 5,786,173 and 5,851,815 and the PCT publication WO 97/35020 under the alternate name, Mch5, are the nucleic acid sequence of caspase 8, nucleic acid fragments thereof including degenerate variants or a full-length complementary sequence thereto and polypeptides encoded by these nucleic acid sequences and fragments (Alnemri et al., 1997; Alnemri et al., 1998; Alnemri et al., 1998).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of caspase 8 and to date, strategies aimed at modulating caspase 8 function have involved the use of antibodies and molecules that block upstream entities such as the death receptors. Consequently, there remains a long felt need for agents capable of effectively inhibiting caspase 8 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of caspase 8 expression.
The present invention provides compositions and methods for modulating caspase 8 expression, including modulation of aberrant forms of caspase 8, including mutated and alternatively spliced forms.